1. Field of the Invention
The present invention relates generally to the fields of diagnostics. More particularly, it concerns the use of methylation-specific PCR in order to identify those individuals having Fragile X syndrome.
2. Description of Related Art
The fragile X syndrome is the most common inherited form of mental retardation and developmental disability. This condition afflicts approximately 1 in 2500 males and 1 in 5000 females. Males with fragile X syndrome usually have mental retardation and often exhibit characteristic physical features and behavior (Hagerman and Silverman, 1992; Warren and Nelson, 1994). Perhaps the most debilitating clinical feature noted in individuals with Fragile X is that of behavior, these characteristics include behavior similar to autism and attention deficit disorder, obsessive-compulsive tendencies, hyperactivity, slow development of motor skills and anxiety fear disorder. When these disabilities are severe and occur simultaneously, the condition is sometimes described as autism, and may be associated with any degree of intelligence. Minorities of individuals with fragile X have autism. Many more have some of the above features. Particularly common in fragile X (and much less so in other conditions) is the combination of a likable, happy, friendly personality with a limited number of autistic-like features such as hand-flapping, finding direct eye contact unpleasant, and some speech and language problems.
Generally speaking, the problems experienced by girls and women with fragile X are similar to those of boys and men. Girls and women with more average intellectual functioning may still have large discrepancies between different ability areas and may show similar concentration problems with impulsiveness, distractibility and difficulty sticking to tasks even if they are not overactive. Shyness and anxiety in social situations can occur.
Fragile X is an X chromosome-linked condition that is characterized by a visible constriction near the end of the X chromosome, at locus q27.3, and there is a tendency for the tip of the X-chromosome to break off under certain conditions in tissue culture. The pattern of inheritance of this condition is atypical of that associated with X-linked conditions. In typical X-linked genetic defects, there is a 50% probability that the male offspring of a female carrier will be afflicted by the defect. Additionally, all males who carry the abnormal gene are afflicted by the X-linked disorder in the typical pattern. Furthermore, since females have two X chromosomes, they normally do not suffer the effects of a single damaged X chromosome. In fragile X syndrome, however, carrier males are phenotypically normal. Certain individuals are carriers of fragile X in that they have a premutation in the FMR1 gene but do not show symptoms of fragile X. Carrier men (transmitting males) pass the premutation to all their daughters but none of their sons. Each child of a carrier woman has a 50% chance of inheriting the gene. The fragile X premutation can be passed silently down through generations in a family before a child is affected by the syndrome. Moreover, about one-third of the females inheriting the fragile X chromosome are afflicted with the disease. Daughters of carrier males are generally non-expressing carriers, but may have afflicted sons. Afflicted daughters occur more frequently among the offspring of carrier mothers than among the offspring of carrier fathers (Brown, 1990).
The genomic region associated with this condition has been identified (Oberle et al., 1991; Kremer et al., 1991; Bell et al., 1991). Researchers have sequenced a cDNA clone derived from this region, called FMR1 (Verkerk et al., 1991). FMR1 has been recognized since 1991 as the gene that causes fragile X (Verkerk et al., 1991; Richards et al., 1991; Eichler et al., 1993; Hirst et al., 1995). The fragile X syndrome is predominantly caused by a large expansion of a CGG trinucleotide repeat in the promoter region of the FMR1 gene, which is associated with methylation and down regulation of transcription. However, it appears that the mutation that ultimately results in the fragile X phenotype occurs in stages. In the early stages, the gene is not fully defective; rather there is a "pre-mutation" of the gene. Carriers of the pre-mutation have a normal phenotype. A further expansion of the premutation occurs in carrier females-that produces the phenotype in their offspring. In individuals who have fragile X syndrome, a defect in FMR1 (a full mutation) of a CGG trinucleotide repeat correlates with methylation of the gene.
Individuals who are not carriers have approximately 30 CGG repeats in their FMR1. Carriers, however, have between about 50 and about 200 CGG repeats. This amplification of the FMR1 CGG sequence is the pre-mutation. Patients with fragile X syndrome have an expansion to the full mutation, which is greater than 200 repeats (Verkerk et al., 1991; Kremer et al., 1991), with as many as several thousand CGG repeats having been reported in afflicted individuals (Oberle et al., 1991). A CpG island, located upstream of the CGG repeat region is methylated when the number of CGG repeats is above a threshold of about 200 copies (Oberle et al., 1991; Kremer et al., 1991, Bell et al., 1991). This methylation results in an inactivation of the gene and silencing of gene transcription which is believed to result in the fragile X phenotype (Verkerk et al., 1991; Oberle et al., 1991; Sutcliffe et al., 1992). Most affected individuals do not express the FMR-1 mRNA (Pieretti et al., 1991). Full mutations also can exist with premutation and normal alleles, and such individuals are known as "mosaic" (Verkerk et al., 1991; Kremer et al., 1991).
A molecular diagnosis of this disorder is based on repeat size and methylation analysis of the FMR1 gene. As methylation has a direct effect on the fragile X phenotype and does not always correlate with repeat expansion, its analysis is an important part of fragile X diagnostics. Molecular testing of the fragile X syndrome is predominantly performed by Southern blot (Rousseau et al., 1991) and/or PCR analysis (Fu et al., 1991; Pergolizzi et al., 1992; U.S. Pat. No. 5,658,764). The advantage of Southern analysis is that methylation status is obtained in addition to repeat expansion. The main disadvantages of this technique are the time taken to perform the procedure, the large amounts of DNA necessary for analysis, and the use of radioisotopes. In PCR-based methods, carriers of the fragile X genotype are identified based on molecular structure of the gene defect. These methods determine whether the number of CGG repeats in the test individual's X-chromosome are characteristic of a normal, carrier or afflicted person. The PCR test, which provides information on repeat size, usually employs radioactivity based assays and has shown limited success in diagnosing full mutations. Other PCR based methods that serve as rapid screening tools for fragile X have been described (Wang et al., 1995; Haddad et al., 1996; Larsen et al., 1997). These methods, however, depend on the non-amplification of a full mutation as an indicator of fragile X and require confirmation by Southern analysis, additionally, these assays are unable to detect a full mutation in the presence of mosaicism.
Additional methods for the diagnosis of fragile X syndrome use microscopy, in which an afflicted individual's chromosomes are examined after cell growth and treatment in tissue culture. The X chromosome is examined to ascertain whether it was characteristically constricted, or had a broken tip. This method is both costly and unreliable. A more recent approach to the identification of fragile X is by assay of FMRP, where a lack of protein is indicative of fragile X (Willemsen et al., 1995). This is a promising technique particularly for large screening studies; however it cannot be used to identify premutations and has a high false negative rate in females.
There currently is no cure for fragile X syndrome, although appropriate education and medications can help maximize the potential of each child. However, most boys and many girls remain significantly affected throughout their lives. The cost to society for treatment, special education, and lost income is staggering. Diagnoses of this syndrome will be helpful in designing appropriate therapies and counseling for affected individuals and carriers of the syndrome. There still exists a need for rapid and reliable assays for Fragile X syndrome to aid those suffering from or carrying the disorder.